Incorporation of unnatural amino acid into human acidic fibroblast growth factor 1 (FGF1) protein with increased thermal, chemical stability and enhanced bioactivity

Abstract

Human acidic fibroblast growth factor 1 (hFGF1) is a critical regulator of cell proliferation, angiogenesis, and tissue regeneration, making it a promising candidate for therapeutic applications in chronic wound healing. However, its clinical utility has been significantly limited by inherent biophysical liabilities, including low thermal stability (Tm ∼42 °C), high susceptibility to proteolytic degradation—particularly cleavage at Arg136 by thrombin—and dependence on heparin for structural integrity and receptor activation. These limitations are especially problematic in diabetic wounds, where elevated protease levels, reduced vascularization, and chronic inflammation compromise the healing environment and degrade native growth factors.

In this study, we engineered a structurally stabilized hFGF1 variant (R136sY) through the site-specific incorporation of sulfotyrosine at position 136, aimed at reversing the positive charge in the heparin-binding pocket to enhance resistance to proteases and improve functional stability. Biophysical characterization confirmed that the R136sY mutant preserved the native β-trefoil architecture while displaying significantly improved thermostability (Tm = 54 °C, a 12 °C increase over wild type hFGF1), chemical stability (Cm = 2.8 M urea vs. 1.5 M for wild type hFGF1), and structural compactness, as evidenced by reduced ANS binding and limited trypsin digestion profiles. Notably, after 40 min of exposure to trypsin, more than 90 % of R136sY remained intact, compared to less than 10 % of hFGF1.

Functionally, R136sY promoted significantly enhanced mitogenic activity in NIH 3T3 fibroblasts at lower concentrations (2–29 ng/mL, p < 0.05) even in the absence of heparin, indicating improved potency relative to hFGF1 (Kerr et al., 2019) 66. has direct translational implications, as it simplifies formulation requirements and ensures activity in protease-rich environments such as chronic diabetic wounds.

Collectively, these findings demonstrate that the R136sY mutant confers critical biophysical and functional improvements by stabilizing the protein's conformation and reducing proteolytic vulnerability. These mechanistic insights provide valuable design principles for engineering second-generation FGF1 analogs optimized for chronic wound care. Specifically, they support the development of protease-resistant, thermostable, and heparin-independent FGF1 variants capable of maintaining biological activity in the harsh microenvironments characteristic of diabetic ulcers—thereby addressing one of the major limitations in current growth factor-based therapies.